Allelic exclusion of the TCRalpha-chain depends on regulation of cell surface levels of TCR, applied differently to one TCRalpha-beta complex than another. It requires signaling through the TCR and the action of the ubiquitin ligase cCbl, indicating that ubiquitination of the TCR is an important factor in the process. Novel fluorescence resonance energy transfer (FRET) microscopy methods will be used to study the dynamics of ubiquitination of TCR in living cells, and to relate this to the process of TCRalpha-chain allelic exclusion. This and conventional assays will be used to analyze endocytosis and recycling of TCR, in order to investigate the pathway and fate of TCR proteins during steady-state endocytosis and recycling, and in TCR down-modulation induced by stimulation of PKC or through activation of TCR ligands. This will allow comparison of how different stimuli can select TCR for degradation versus recycling and will be used to study the post-translation allelic exclusion of TCR alpha-chain. A transgenic mouse strain with two different rearranging TCRalpha-chain miniloci, each with a different Valpha-region, will be used to study of the way in which thymocytes or peripheral T cells that can express two alpha-chains regulate the cell surface level of the two alpha-beta complexes, such that mature cells typically express only one combination on the cell surface. Intracellular fates of the two alpha-chains during thymocyte selection will be determined, such as recycling, ubiquitination and degradation. Fluorescent TCRalpha-chain chimeras will be used to study allelic exclusion, comparing endocytosis, recycling, ubiquitination and degradation in living transfected T cells subjected to different stimuli. PKCeta is induced during thymocyte positive selection. Like PKCtheta, it is recruited to the immunological synapse in thymocytes. Because it is over-expressed, and expressed much earlier than normal, in PKCtheta knockout thymus, its potential role in thymocyte development and T cell activation will be determined, using fluorescence microscopy, shRNA and knockout mice.